Intraspinally administered alpha2-adrenergic agonists represent a new approach to pain management. Epidurally administered clonidine produces profound analgesia, free of respiratory depression, in patients following surgery and in cancer patients tolerant to opioids. However, in analgesic doses clonidine also decreases blood pressure and causes sedation. Preliminary work suggests that alpha2-adrenergic agonists produce analgesia by activating cholinergic neurons in the spinal cord, and that spinally applied alpha2-adrenergic and cholinergic agonists have opposing actions on blood pressure, such that activation of both may produce profound analgesia with minimal hemodynamic depression. In this continuation proposal, we focus on interactions between alpha2-adrenergic and cholinergic systems, utilizing 3 approaches. First, the detailed anatomy of the sheep spinal cord dorsal horn and intermediolateral cell column (IML) will be defined, including the relationship between alpha2-adrenergic receptors and cholinergic neurons, and location of muscarinic receptor subtypes. Second, alpha2-adrenergic influences on acetylcholine (ACh) release into cerebrospinal fluid (CSF) will be examined. Bulbospinal noradrenergic antinociceptive pathways are naturally activated by noxious stimuli, causing release of norepinephrine (NE) in CSF. A detailed pharmacologic examination of coincident release of NE and ACh in CSF will be performed in anesthetized ewes receiving noxious electrical stimulation. Third, the interaction between spinally administered alpha2-adrenergic and cholinergic agonists and antagonists and other drugs acting on cholinergic systems on integrated responses (antinociception, blood pressure, heart rate) will be described in unanesthetized ewes. Together these studies will define the anatomic, physiologic, and pharmacologic basis of interactions between spinal alpha2-adrenergic and cholinergic systems in analgesia and cardiovascular regulation. The proposed studies are likely to provide new information relevant to mechanisms of spinal analgesia and to improved clinical care.